The term angiogenesis refers to the generation or formation of new blood vessels into a tissue or organ. Angiogenesis can occur both during some physiological processes and/or in some pathological conditions. For example, angiogenesis can be seen to occur during wound healing, fetal growth, corpus luteum, and endometrium, etc., (1). Endothelial cells, which cause to form the inner lining of the blood vessels, are constituted by a thin layer of epithelial cells and these cells are necessary for the process of angiogenesis. During the process of angiogenesis, irrespective of whether it is physiological or pathological, the endothelial cells release enzymes which can produce erosions of the basement membrane through which the endothelial cells cause protrusions. In response to the stimuli given by various agents, endothelial cells proliferate and migrate through the protrusions and form a sprout of the parent blood vessel. These endothelial cell sprouts can merge to form capillary loops leading to the formation of new blood vessel(s). If the blood vessels are in a tumor area, these new blood vessels in turn will provide enough nutrients and energy sources so that tumor cells can divide, proliferate and grow both in number and size.
Thus, the process of angiogenesis is both essential and critical to the growth of cancer. The other pathological states in which angiogenesis plays a critical role include: rheumatoid arthritis, psoriasis, scleroderma, myocardial angiogenesis, corneal diseases, diabetic retinopathy associated with neovascularization, macular degeneration, ovulation, menstruation etc. The process of angiogenesis also appears to be critical for tumor metastasis.
Since angiogenesis is such a critical process in the promotion of cancer and tumor metastasis, several researches have been trying to devise methods or develop drugs which can selectively suppress angiogenesis with the hope that this would eventually lead to the inhibition of tumor growth. There are other situations where uncontrolled angiogenesis is undesirable. For instance, formation of new blood vessels in an area like cornea during the process of healing of the corneal ulcer, if it is in excess, can lead to corneal scar formation.
In the case of rheumatoid arthritis, angiogenesis can lead to continued inflammation in the joints and also to osteoporosis. In such an instance, prevention of formation of new blood vessels will lead to reduction in inflammation and also prevention of fibrous ankylosis and bony ankylosis. Thus, selective prevention and control of angiogenesis may be of benefit in the aforementioned conditions, as well as in several other conditions such as: uterine fibroids, psoriasis, scleroderma, diabetic retinopathy, keloids, ovulation etc. Another area where prevention of angiogenesis will be of benefit is in the inhibition of ovulation and menstruation and growth of placenta and this will lead to prevention of fertilization and growth of the fetal tissue. This may, thus, form a new approach in the development of fertility control measures.
Two naturally occurring molecules which have been identified to adversely influence or inhibit angiogenesis are ANGIOSTATIN® and ENDOSTATIN® (2). Both these molecules are proteins. ANGIOSTATIN is a protein of molecular weight approximately 38 kD and has an amino acid sequence substantially similar to that of a fragment of murine plasminogen beginning at amino acid number 98 of an intact murine plasminogen molecule. The amino acid sequence of ANGIOSTATIN varies only slightly between species. The amino acid sequence of the human ANGIOSTATIN is substantially similar to the murine plasminogen fragment. But, it may be mentioned here that the active human ANGIOSTATIN sequence starts either at the amino acid number 97 or 99 of an intact human plasminogen amino acid sequence. In addition, human plasminogen has potent anti-angiogenic activity even in a mouse tumor model. This explains why both murine and human plasminogens and ANGIOSTATIN/ENDOSTATIN molecules show fairly similar anti-angiogenic activities in a variety of animal tumor models (3).
U.S. Pat. No. 5,792,845 issued on Aug. 11, 1998 to O'Reilly et al, teaches that therapies directed at control of the angiogenic process could lead to the abrogation or mitigation of certain diseases. O'Reilly et al suggests that modulation of the formation of capillaries in angiogenic processes (such as wound healing and reproduction) is useful since undesired and uncontrolled angiogenesis can cause certain diseases to progress. O'Reilly et al teaches that ANGIOSTATIN protein has the capability of inhibiting angiogenesis, eg., to inhibit the growth of bovine capillary endothelial cells in culture in the presence of fibroblast growth factor.
U.S. Pat. No. 5,792,845 issued on Aug. 11, 1998 to O'Reilly et al, teaches that therapies directed at control of the angiogenic process could lead to the abrogation or mitigation of certain diseases. O'Reilly et al suggests that modulation of the formation of capillaries in angiogenic processes (such as wound healing and reproduction) is useful since undesired and uncontrolled angiogenesis can cause certain diseases to progress. O'Reilly et al teaches that ANGIOSTATIN protein has the capability of inhibiting angiogenesis, e.g., to inhibit the growth of bovine capillary endothelial cells in culture in the presence of fibroblast growth factor.
U.S. Pat. No. 5,932,545 issued on Aug. 3, 1999 to Henkin et al teaches an anti-angiogenic drug in the form of a peptide or a salt thereof, to treat cancer, arthritis and retinopathy. The Henkin et al patent states however that angiogenesis inhibitors could cause systemic toxicity in humans. ANGIOSTATIN in the O'Reilly patent '845 is described and claimed as an Isolated nucleotide molecule with a specific sequence. It has been stated however that the ANGIOSTATIN molecule as known at present is not suitable for clinical trials.
ENDOSTATIN, which is also similar to ANGIOSTATIN, has been shown to cause a dramatic reduction of primary and metastatic tumors in experimental animals. ENDOSTATIN is a 20 kDa C-terminal fragment of collagen XVIII. ENDOSTATIN could specifically inhibit endothelial cell proliferation and angio-genesis and thus, block tumor growth (2, 4).
It is important to note that ANGIOSTATIN is derived from plasminogen or plasmin. It has been shown that human prostate carcinoma cell lines express enzymatic activity that can generate bioactive ANGIOSTATIN from purified human plasminogen or plasmin. This bioactive ANGIOSTATIN has been shown to inhibit human endothelial cell proliferation, basic fibroblast growth factor-induced migration, endothelial cell tube formation, and basic fibroblast growth factor-induced corneal angiogenesis. In an extension of this study, it was noted that a serine proteinase is necessary for ANGIOSTATIN generation (5).
ANGIOSTATIN, derived from plasminogen, selectively inhibits endothelial cell proliferation. When ANGIOSTATIN is given systemically it shows potent inhibitory action on the growth of tumor and renders metastatic and primary tumors to go into a dormant state by striking a balance between the rate of proliferation and apoptosis of the tumor cells (6). The very identification of ANGIOSTATIN has come from the observation that when a primary tumor is present, the growth of metastases is suppressed. On the other hand, after tumor removal, the previously dormant metastases develop new blood vessels (neovascularization) and grow. Both serum and urine from the tumor-bearing animals, but not from controls, showed very specific inhibitory action on the growth of endothelial cells. Subsequent studies led to the purification of this inhibitor of endothelial cells which was later identified as a 38 kD plasminogen fragment namely ANGIOSTATIN. It is now known that ANGIOSTATIN, which can also be obtained by a limited proteolytic digestion of human plasminogen, but not intact plasminogen can be administered systemically to block neovascularization and growth of metastases and primary tumors. A recombinant human ANGIOSTATIN which comprises of kringles 1-4 of human plasminogen (amino acids 93-470) expressed in Pichia pastoris has been prepared and is now available for use. This recombinant ANGIOSTATIN showed the same physical properties as that of the natural ANGIOSTATIN in terms of molecular size, binding to lysine, reactivity with antibody to kringles 1-3 (3, 7). This recombinant ANGIOSTATIN when given to experimental animals, showed anti-angiogenic and anti-tumor activity (3). In addition, recombinant mouse ANGIOSTATIN was produced using the baculo-virus infected insect cells (8), which also (the secreted protein) showed potent inhibitory action on the proliferation of bovine capillary endothelial cells in vitro. The conversion of plasminogen to ANGIOSTATIN by PC-3 cells is now identified to be due to two components released, urokinase (uPA) and free sulfhydryl donors (FSDs). This is supported by the fact that even in a cell-free system, ANGIOSTATIN can be generated from plasminogen by plasminogen activators (u-PA, tissue-type plasminogen activator, tPA or streptokinase) in combination with any one of free sulfhydryl donors such as N-acetyl-L-cysteine, D-penicillamine, captopril, L-cysteine, or reduced glutathione. This cell-free derived ANGIOSTATIN also showed anti-angiogen activity both in vitro and in vivo and suppressed the growth of Lewis lung carcinoma metastases (9).
ANGIOSTATIN administration to mice with subcutaneous hemangioendothelioma and associated disseminated intravascular coagulopathy revealed that in addition to a significant reduction in the size of the tumor, increased survival, decrease in thrombocytopenia and anemia was noted (10). This indicates that ANGIOSTATIN may also be useful to treat disseminated intravascular coagulopathy.
One of the mechanisms by which ANGIOSTATIN inhibits endothelial cell proliferation includes its ability to affect by 4 to 5 fold the expression of E-selectin in proliferating endothelial cells (11). On the other hand, ANGIOSTATIN did not alter cell cycle progression significantly. Further, ANGIOSTATIN also enhanced the adhesion activity in proliferating endothelial cells.
Rivas et al (12) studied the possible relationship between human macropahge metalloelastase (HME) expression, a member of the human matrix metalloproteinase family, which is believed to play an important role in ANGIOSTATIN generation, and ANGIOSTATIN production. Their study showed that patients whose tumors did not express HME mRNA and so did not produce ANGIOSTATIN, had poorer survival than those whose tumors showed high expression of HME mRNA and ANGIOSTATIN generation. This study suggests that HME gene expression is closely associated with ANGIOSTATIN generation and prognosis in patients with hepatocellular carcinoma (HCC). This relationship between HME and ANGIOSTATIN is understandable since, metalloproteinase(s) can block angiogenesis by converting plasminogen to ANGIOSTATIN (12,13,14).
Another mechanism by which recombinant human and murine ANGIOSTATINs can block angiogenesis is by inducing apoptosis (programmed cell death) of endothelial cells (15), similar to that seen with tumor necrosis factor (TNF) and transforming factor-beta 1 (TGF-beta1), which are also known to induce apoptosis in endothelial cells.
Yet another mechanism by which ANGIOSTATIN can produce apoptosis and inhibit angiogenesis is probably by binding to ATP synthase. Using human umbilical endothelial vein endothelial cells, Moser et al (16) observed that ANGIOSTATIN bound in a concentration-dependent, saturable manner to the alpha/beta sub-units of ATP synthase. This binding of ANGIOSTATIN to the alpha/beta sub-unit of ATP synthase was inhibited by as much as 90% in the presence of anti-alpha-sub-unit ATP synthase antibody. This indicates that ANGIOSTATIN by binding to ATP synthase may actually shut-off ATP synthesis in the endothelial cells and this would eventually lead to death of the cells due to the non-availability of ATP, the main energy source for the survival of the cells. In addition, it was also reported that ANGIOSTATIN can inhibit extra-cellular-matrix-enhanced, t-PA catalysed plasminogen activation. This results in reduced invasive activity of endothelial cells (17). All these results indicate that ANGIOSTATIN has multiple actions by which it is able to block endothelial cell proliferation and angiogenesis.
Some of the factors which are known to inhibit the generation of ANGIOSTATIN include TGF-beta1 and plasminogen activator inhibitor type-1 (PAI-1), at least, by human pancreatic cancer cells in vitro (18).
Twining et al (19) showed that plasmin, the active form of plasminogen, is necessary for the maintenance of normal cornea and for corneal wound healing. It was also noted that plasmin is a major serine proteinase in the human cornea and that cornea can synthesize plasminogen. Both interleukin-1alpha and 1 beta stimulated corneal plasminogen synthesis by almost 2 to 3fold where as interleukin-6 decreased corneal plasminogen synthesis by 40%. Thus, cornea seems to have the ability to synthesize plasminogen, the precursor of plasmin and ANGIOSTATIN, and also regulate its synthesis in response to injury and inflammation and IL-1 and IL-6 (19).
Though both ANGIOSTATIN and ENDOSTATIN and other similar anti-angiogenic molecules provided an important therapeutic advance for cancer treatment, it should be emphasized here that the needed dosages of these proteins, especially ANGIOSTATIN used in the animal studies seem to be too high for clinical trials (20). Further, repeated injections and long-term treatment with ANGIOSTATIN are required to obtain its maximal anti-tumor effect. In view of this, methods to supplement the anti-angiogenic action of ANGIOSTATIN and ENDOSTATIN and other similar compounds are considered desirable. These methods include: use of ANGIOSTATIN along with other conventional anti-cancer drugs including radiation and novel methods of delivery of ANGIOSTATIN to tumor cells (21). Mauceri et al (22) studied the combined effect of radiation with ANGIOSTATIN and showed that this combination produced no increase in toxicity towards normal tissue. Both in vitro and in vivo studies showed that these agents (radiation and ANGIOSTATIN) in combination target the tumor vasculature. In an extension of this study, Gorski et al (23) demonstrated that the efficacy of experimental radiation therapy is potentiated by brief concomitant exposure of the tumor vasculature to ANGIOSTATIN.
Two novel methods of delivery of ANGIOSTATIN and similar compounds to the tumor cells that have been tried include:                (a) Nguyen et al (24) generated recombinant adeno-associated virus (rAAV) vectors that carry genes encoding for ANGIOSTATIN, ENDOSTATIN, and an antisense mRNA species against vascular endothelial growth factor (VEGF). These rAAVs efficiently transduced three human tumor cell lines that have been tested. Further, testing of the conditioned media from cells transduced with this rAAV or with rAAV-expressing ENDOSTATIN or ANGIOSTATIN inhibited effectively endothelial cell proliferation in vitro. These results indicate that rAAVs can be used to block angiogenesis and cancer growth.        (b) In a different approach, Chen et al (25) examined whether liposomes complexed to plasmids encoding ANGIOSTATIN or ENDOSTATIN can inhibit angiogenesis and growth of tumors. These studies revealed that plasmids expressing ANGIOSTATIN (PCI-angio) or ENDOSTATIN (PCI-endo) can effectively reduce angiogenesis and the size of the tumors implanted in the mammary fat pad of male mice to a significant degree. In addition, liposomes complexed to PCI-endo when given intravenously reduced tumor growth in nude mice by nearly 40% when compared to controls (25).        